U.S. patent application number 09/946789 was filed with the patent office on 2002-05-30 for reverse-turn mimetics and methods relating thereto.
This patent application is currently assigned to Molecumetics ltd.. Invention is credited to Kahn, Michael, Stasiak, Marcin.
Application Number | 20020065416 09/946789 |
Document ID | / |
Family ID | 23535809 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020065416 |
Kind Code |
A1 |
Stasiak, Marcin ; et
al. |
May 30, 2002 |
Reverse-turn mimetics and methods relating thereto
Abstract
Conformationally constrained compounds which mimic the secondary
structure of reverse-turn regions of biologically active peptides
and proteins having the following structure are disclosed: 1
wherein A, R.sub.1, R.sub.2, R.sub.2a, R.sub.3, R.sub.3a and
R.sub.4 are as defined herein. Such compounds have utility over a
wide range of fields, including use as diagnostic and therapeutic
agents. In particular, compounds of this invention are useful in
pharmaceutical compositions as anti-inflammatory agents. Libraries
containing the compounds of this invention are also disclosed, as
well as methods for screening the same to identify biologically
active members.
Inventors: |
Stasiak, Marcin; (Kirkland,
WA) ; Kahn, Michael; (Kirkland, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Molecumetics ltd.
Bellevue
WA
|
Family ID: |
23535809 |
Appl. No.: |
09/946789 |
Filed: |
September 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09946789 |
Sep 4, 2001 |
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09388854 |
Sep 1, 1999 |
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6294525 |
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Current U.S.
Class: |
544/350 ;
530/331; 540/568 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; G01N 2333/70546 20130101; A61P 13/12 20180101;
A61P 25/00 20180101; C07D 487/04 20130101; A61P 9/08 20180101; A61P
27/02 20180101; A61P 25/28 20180101; A61K 38/00 20130101; A61P
19/02 20180101; A61P 3/10 20180101; A61P 1/00 20180101; A61P 9/10
20180101; A61P 17/00 20180101; C07K 5/06139 20130101; A61P 11/06
20180101; C40B 40/00 20130101; A61P 11/00 20180101; A61P 29/00
20180101; A61P 37/08 20180101 |
Class at
Publication: |
544/350 ;
530/331; 540/568 |
International
Class: |
C07D 487/02; C07K
005/00 |
Claims
We claim:
1. A compound having the structure: 295and pharmaceutically
acceptable salts and stereoisomers thereof, wherein A is
--(CR.sub.5R.sub.5a).sub.n-- - where n is 1, 2 or 3; R.sub.2,
R.sub.3 and R.sub.5 are, at each occurrence, the same or different
and independently an amino acid side chain moiety or amino acid
side chain derivative, a peptide or peptide derivative, a linker or
a solid support; R.sub.2a, R.sub.3a and R.sub.5a are, at each
occurrence, the same or different and independently hydrogen,
hydroxy, --COOH, --CONH.sub.2, --R.sub.6, --OR.sub.6, --COOR.sub.6,
--COR.sub.6 or --CONHR.sub.6, where R.sub.6 is lower alkyl
optionally substituted with halogen or hydroxy; and R.sub.1 and
R.sub.4 represent the remainder of the molecule, with the proviso
that when R.sub.1 is --C(.dbd.O)OMe and R.sub.4 is benzyl, R.sub.2
is not isopropyl when R.sub.3 is methyl and R.sub.2 is not methyl
when R.sub.3 is isopropyl.
2. The compound of claim 1 wherein R.sub.2a and R.sub.3a are
hydrogen, and the compound has the structure: 296
3. The compound of claim 2 wherein n is 1, R.sub.5a is hydrogen,
and the compound has the structure: 297
4. The compound of claim 3 wherein R.sub.5 is hydrogen.
5. The compound of claim 2 wherein n is 2, R.sub.5a is hydrogen at
each occurrence, and the compound has the structure: 298
6. The compound of claim 5 wherein R.sub.5 is hydrogen at each
occurrence, and the compound has the structure: 299
7. The compound of claim 1 wherein R.sub.3a is hydrogen, and the
compound has the structure: 300
8. The compound of claim 2 or 7 wherein R.sub.2, R.sub.3 and
R.sub.4 are the same or different and are independently an amino
acid side chain moiety or an amino acid side chain derivative.
9. The compound of claim 1 wherein R.sub.1 is --C(.dbd.O)OR.sub.7,
--C(.dbd.O)NHR.sub.7 or --SO.sub.2R.sub.7 where R.sub.7 is an amino
acid side chain moiety or an amino acid side chain derivative.
10. The compound of claim 9 wherein R.sub.7 aryl or arylalkyl
optionally substituted with halogen, --OH, --COOH, --NH.sub.2 or
alkyl.
11. The compound of claim 9 wherein R.sub.2a and R.sub.3a are
hydrogen.
12. The compound of claim 9 wherein R.sub.2 is
--(CH.sub.2).sub.2COOH.
13. The compound of claim 9 wherein R.sub.3 is benzyl or
substituted benzyl.
14. The compound of claim 9 wherein R.sub.1 is
--SO.sub.2R.sub.7.
15. The compound of claim 14 wherein the compound has the
structure: 301
16. The compound of claim 14 wherein the compound has the
structure: 302
17. The compound of claim 14 wherein the compound has the
structure: 303
18. The compound of claim 14 wherein the compound has the
structure: 304
19. The compound of claim 14 wherein the compound has the
structure: 305
20. The compound of claim 14 wherein the compound has the
structure: 306
21. The compound of claim 14 wherein the compound has the
structure: 307
22. The compound of claim 14 wherein the compound has the
structure: 308
23. The compound of claim 14 wherein the compound has the
structure: 309
24. A composition comprising a compound of claim 1 and a
pharmaceutically acceptable carrier.
25. A library of compounds comprising a plurality of library
members, wherein at least one library member is a compound of claim
1.
26. A method of identifying a biologically active compound,
comprising screening the library of compounds of claim 25 for
biological activity.
27. A method for treating an inflammatory or cell adhesion-mediated
disease comprising administering to a warm-blooded animal in need
thereof an effective amount of the composition of claim 24.
28. The method of claim 27 wherein the disease is an inflammatory
disease.
29. The method of claim 27 wherein the disease is a cell
adhesion-mediated disease.
30. The method of claim 27 wherein the disease is rheumatoid
arthritis, Alzheimer's disease, AIDS dementia, ARDS, asthma,
allergies, inflammatory bowel disease, CNS inflammation, atopic
dermatitis, encephalitis, multiple sclerosis, meningitis,
nephritis, retinitis or psoriasis.
31. The method of claim 27 wherein the disease is type I diabetes,
atherosclerosis, myocardial ischemia, restenosis, stroke or tumor
metastasis.
Description
TECHNICAL FIELD
[0001] The present invention relates generally to reverse-turn
mimetics, including inhibitors of cell adhesion-mediated disease,
as well as to a chemical library of reverse-turn mimetics.
BACKGROUND OF THE INVENTION
[0002] In the search for new therapeutics, the pharmaceutical
industry has increasingly turned to the techniques of combinatorial
chemistry, parallel synthesis, and high throughput screening to
generate and optimize lead compounds (Combinatorial Chemistry and
Molecular Diversity in Drug Discovery Gordon and Kerwin, Eds., John
Wiley & Sons, New York, 1998; The Combinatorial Index Bunin,
Academic Press, New York, 1998; A Practical Guide to Combinatorial
Chemistry Czarnik and DeWitt, Eds., American Chemical Society,
Washington, DC, 1997; High Throughput Screening: The Discovery of
Bioactive Substances Devlin, Marcel Dekker, New York, 1997). These
techniques can produce libraries of hundreds to hundreds of
thousands--or more--of compounds in a short period of time. The
libraries are then assayed against targets of interest, often in a
highly automated fashion, to identify biologically active
compounds. Libraries, which are simply collections of compounds,
may be tightly focused around a specific template or contain a
variety of unrelated templates. In many instances, the diversity of
the library is an important design parameter.
[0003] On a basic level, the number of points of diversity on a
molecular template or scaffold, i.e., the number of positions at
which variation in structure may be introduced, has a practical
effect on the ease with which large libraries may be created. When
combinatorial techniques are employed, a template that contains
three points of diversity would give rise to 8000 compounds if 20
components are used to derivatize each point and a total of 60
reactions are carried out (20.sup.3). However, a template with four
points of diversity will yield over 50,000 compounds when 15
components are used at each point in a total of 60 reactions
(15.sup.4). In general, large libraries may be created more
efficiently on templates allowing more possibilities for
derivatization.
[0004] In order to increase the chances of finding a biologically
active compound for a particular target, it is usually desirable to
synthesize a library spanning a range of both conformational space
and chemical properties such as hydrophobicity and hydrogen bonding
ability. At the same time, low molecular weight is often a goal as
well, since compounds of less than 500 Daltons are perceived as
more likely to have favorable pharmacokinetic properties in
relation to higher molecular weight compounds. All these
characteristics point to the continuing need for small compact
templates that support a wide range of substituents and which are
simple to synthesize.
[0005] Reverse-turns comprise one of three classes of protein
secondary structure and display three (gamma-turn), four
(beta-turns), or more (loops) amino acid side chains in a fixed
spatial relationship to each other. Turns have proven important in
molecular recognition events (Rose et al., Advances in Protein
Chemistry 37:1-109, 1985) and have engendered a burgeoning field of
research into small molecule mimetics of them (e.g., Hanessian et
al., Tetrahedron 53:12789-12854, 1997). Many mimetics have either
been external turn-mimetics which do not allow for the display of
all the physiologically relevant side-chains (e.g., Freidinger et
al., Science 210:656-8, 1980) or small, conformationally mobile
cyclic peptide derivatives (e.g., Viles et al., Eur. J. Biochem.
242:352-62, 1996). However, non-peptide compounds have been
developed which closely mimic the secondary structure of
reverse-turns found in biologically active proteins or peptides.
For example, U.S. Pat. Nos. 5,475,085, 5,670,155 and 5,672,681 to
Kahn and published PCT WO94/03494 to Kahn all disclose
conformationally constrained, non-peptidic compounds which mimic
the three-dimensional structure of reverse-turns. More recently,
U.S. Pat. No. 5,929,237 to Kahn, and published PCT WO97/15577 to
Kahn and PCT WO98/49168 to Kahn et al. disclosed additional, highly
constrained bicyclic heterocycles as reverse-turn mimetics.
Nevertheless, as no one template can mimic every type of turn,
there remains a need in the art for additional reverse-turn
templates.
[0006] Cell adhesion is critical to the viability of living
organisms. Adhesion holds multicellular tissues together and
directs embryonic development. It plays important roles in wound
healing, eradication of infection and blood coagulation. Integrins
are a family of cell surface proteins intimately involved in all of
these functions. They have been found in nearly every type of human
cell except red blood cells. Abnormalities in integrin function
contribute to a variety of disorders including inflammatory
diseases, heart attack, stroke, and cancer.
[0007] Integrins consist of heterodimers of .alpha. and .beta.
subunits, non-covalently bound to each other. These cell surface
receptors extend through the cell membrane into the cytoplasm. At
least 15 different .alpha. and 9 different .beta. subunits are
known. However, because most .alpha. proteins associate with only a
single .beta. there are about 21 known integrin receptors. On the
cell surface the heads of the two subunits contact each other to
form a binding surface for extracellular protein ligands, allowing
attachment to other cells or to the extracellular matrix. The
affinity of these receptors may be regulated by signals from
outside or within the cell. For example, recruitment of leukocytes
to the site of injury or infection involves a series of adhesive
interactions. Weak interaction between endothelial and leukocyte
selectins and carbohydrates mediate transient adhesion and rolling
of the leukocyte along the vessel wall. Various chemokines and
other trigger factors released by the site of inflammation serve as
signals to activate integrins from a quiescent to a high affinity
state. These activated integrins then bind their cognate ligands on
the surface of the endothelial cells, resulting in strong adhesion
and flattening of the leukocyte. Subsequently the leukocyte
migrates through the endothelium into the tissue below.
[0008] Integrin .alpha..sub.4.beta..sub.1 mediates cell adhesion
primarily through binding to either vascular cell adhesion
molecule-1 (VCAM-1) or an alternatively spliced variant of
fibronectin containing the type III connecting segment (IIICS). A
variety of cells involved in inflammation express
.alpha..sub.4.beta..sub.1, including lymphocytes, monocytes,
basophils and eosinophils, but not neutrophils. Monoclonal
antibodies to the .alpha..sub.4 subunit have been used to validate
.alpha..sub.4-containing integrins as potential therapeutic targets
in animal models of rheumatoid arthritis (Barbadillo et al.,
Springer Semin Immunopathol. 16:427-36, 1995; Issekutz et al.,
Immunology 88:569-76, 1996), acute colitis (Podolsky et al., J.
Clin. Invest. 92:372-80, 1993), multiple sclerosis (Yednock et al.,
Nature 356:63-6, 1992), asthma (Abraham et al., J. Clin. Invest.
93:776-87, 1994) U.S. Pat. No. 5,871,734) and diabetes (Tsukamoto
et al., Cell Immunol. 165:193-201, 1995). More recently, low
molecular weight peptidyl derivatives have been produced as
competitive inhibitors of .alpha..sub.4.beta..sub.1 and one has
been shown to inhibit allergic airway responses in sheep (Lin et
al., J. Med. Chem. 42:920-34, 1999).
[0009] It has been shown that a key sequence in IIICS involved in
binding to .alpha..sub.4.beta..sub.1 is the 25 residue peptide CS1,
and within that sequence the minimally recognized motif is the
tripeptide, LDV. A similar sequence, IDS, has been implicated in
the binding of VCAM-1 to .alpha..sub.4.beta..sub.1. X-ray crystal
structures of an N-terminal two-domain fragment of VCAM-1 show that
the IDS sequence is part of an exposed loop linking two
beta-strands (Jones et al., Nature 373:539-44, 1995; Wang et al.,
Proc. Natl. Acad. Sci. USA 92:5714-8, 1995). Cyclic peptides and
derivatives thereof which adopt reverse-turn conformations have
proven to be inhibitors of VCAM-1 binding to
.alpha..sub.4.beta..sub- .1 (WO 96/00581; WO 96/06108; Doyle et
al., Int. J. Pept. Protein Res. 47:427-36, 1996). In addition, a
number of potent and selective (versus .alpha..sub.5.beta..sub.1)
cyclic peptide-based inhibitors have been discovered (Jackson et
al., J. Med. Chem. 40:3359-68, 1997). Several non-peptidyl
beta-turn mimetics have also been reported to bind
.alpha..sub.4.beta..sub.1 with IC.sub.50s in the low micromolar
range (Souers et al., Bioorg. Med. Chem. Lett. 8:2297-302, 1998).
Numerous phenylalanine and tyrosine derivatives have also been
disclosed as inhibitors of .alpha..sub.4.beta..sub.1 (WO 99/06390;
WO 99/06431; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO
99/06437; WO 98/54207; WO 99/10312; WO 99/10313; WO 98/53814; WO
98/53817; WO 98/58902). However, no potent and orally available
small molecule inhibitors have been disclosed.
[0010] A related integrin, .alpha..sub.4.beta..sub.7, is expressed
on the surface of lymphocytes and binds VCAM-1, fibronectin and
mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Integrin
.alpha..sub.4.beta..sub.7 and MAdCAM mediate recirculation of a
subset of lymphocytes between the blood, gut, and lymphoid tissue.
Similar to VCAM-1 and Fibronectin CS-1 there is a tripeptide
sequence, LDT, present on the CD loop of MAdCAM-1 which is
important for recognition by .alpha..sub.4.beta..sub.7. An X-ray
crystal structure shows this sequence is also part of a turn
structure (Tan et al., Structure 6:793-801, 1998). Recent studies
have shown that .alpha..sub.4.beta..sub.7 may also play a part in
diseases such as asthma (Lobb et al., Ann. NY Acad. Sci.
796:113-23, 1996), inflammatory bowel disease (Fong et al.,
Immunol. Res. 16:299-311, 1997), and diabetes (Yang et al.,
Diabetes 46:1542-7, 1997). In addition, while .alpha..sub.4
integrins appear to be down-regulated in carcinomas such as
cervical and prostate, they appear to be up-regulated in metastatic
melanoma (Sanders et al., Cancer Invest. 16:329-44, 1998),
suggesting that inhibitors of .alpha..sub.4.beta..sub.1 and
.alpha..sub.4.beta..sub.7 may be useful as anticancer agents.
[0011] While significant advances have been made in the synthesis
and identification of conformationally constrained, reverse-turn
mimetics, there is still a need in the art for small molecules that
mimic the secondary structure of peptides. There is also a need in
the art for libraries containing such members, particularly those
small templates capable of supporting a high diversity of
substituents. In addition, there is a need in the art for
techniques for synthesizing these libraries and screening the
library members against biological targets to identify bioactive
library members. Further, there is a need in the art for small,
orally available inhibitors of integrins, for use in treating
inflammatory diseases and cardiovascular diseases, as well as some
cancers. In particular there is a need for inhibitors of
.alpha..sub.4.beta..sub.1 and .alpha..sub.4.beta..sub.7, for use in
the treatment of rheumatoid arthritis, asthma, diabetes and
inflammatory bowel disease.
[0012] The present invention fulfills these needs, and provides
further related advantages.
SUMMARY OF THE INVENTION
[0013] In brief, the present invention is directed to
conformationally constrained compounds which mimic the secondary
structure of reverse-turn regions of biologically active peptides
and proteins (also referred to herein as "reverse-turn mimetics").
The compounds of the present invention have the following general
structure (I): 2
[0014] including pharmaceutically acceptable salts and
stereoisomers thereof, wherein A and R.sub.1 through R.sub.4 are as
defined below.
[0015] The present invention is also directed to libraries
containing compounds of structure (I), as well as methods for
synthesizing such libraries and methods for screening the same to
identify biologically active compounds. In addition, compositions
containing a compound of this invention in combination with a
pharmaceutically acceptable carrier are disclosed. Methods of use
for treating cell-adhesion-mediated disease with the compounds of
this invention and compositions comprising them are also
disclosed.
[0016] These and other aspects of this invention will be apparent
upon reference to the following detailed description. To this end,
various references are set forth herein which describe in more
detail certain procedures, compounds and/or compositions, and are
incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is directed to reverse-turn mimetics
and chemical libraries containing reverse-turn mimetics. The
reverse-turn mimetics of the present invention are useful as
bioactive agents, including (but not limited to) use as diagnostic,
prophylactic and/or therapeutic agents, especially as
anti-inflammatory agents. The reverse-turn mimetic libraries of
this invention are useful in the identification of such bioactive
agents. In the practice of the present invention, the libraries may
contain from tens to hundreds to thousands (or greater) of
individual reverse-turn mimetics (also referred to herein as
"members").
[0018] In one aspect of the present invention, a reverse-turn
mimetic is disclosed having the following structure (I): 3
[0019] and pharmaceutically acceptable salts and stereoisomers
thereof,
[0020] wherein
[0021] A is --(CR.sub.5R.sub.5a).sub.n-- where n is 1, 2 or 3;
[0022] R.sub.2, R.sub.3 and R.sub.5 are, at each occurrence, the
same or different and independently an amino acid side chain moiety
or amino acid side chain derivative, a peptide or peptide
derivative, a linker or a solid support;
[0023] R.sub.2a, R.sub.3a and R.sub.5a are, at each occurrence, the
same or different and independently hydrogen, hydroxy, --COOH,
--CONH.sub.2, --R.sub.6, --OR.sub.6, --COOR.sub.6, --COR.sub.6 or
--CONHR.sub.6, where R.sub.6 is lower alkyl optionally substituted
with halogen or hydroxy; and
[0024] R.sub.1 and R.sub.4 represent the remainder of the molecule,
with the proviso that when R.sub.1 is --C(.dbd.O)OMe and R.sub.4 is
benzyl, R.sub.2 is not isopropyl when R.sub.3 is methyl and R.sub.2
is not methyl when R.sub.3 is isopropyl.
[0025] As used herein, an "amino acid side chain moiety" represents
any amino acid side chain moiety present in naturally occurring
proteins including (but not limited to) the naturally occurring
amino acid side chain moieties identified in Table 1 below. Other
naturally occurring amino acid side chain moieties of this
invention include (but are not limited to) the side chain moieties
of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine,
.gamma.-carboxyglutamate, phosphotyrosine and phosphoserine. In
addition, glycosylated amino acid side chains may also be used in
the practice of this invention, including (but not limited to)
glycosylated threonine, serine and asparagine.
1TABLE 1 AMINO ACID SIDE CHAIN MOIETIES Amino Acid Side Chain
Moiety Amino Acid --H Glycine --CH.sub.3 Alanine
--CH(CH.sub.3).sub.2 Valine --CH.sub.2CH(CH.sub.3).sub.2 Leucine
--CH(CH.sub.3)CH.sub.2C- H.sub.3 Isoleucine
--(CH.sub.2).sub.4NH.sub.2 Lysine
--(CH.sub.2).sub.3NHC(NH.sub.2)NH.sub.2 Arginine 4 Histidine
--CH.sub.2COOH Aspartic acid --CH.sub.2CH.sub.2COOH Glutamic acid
--CH.sub.2CONH.sub.2 Asparagine --CH.sub.2CH.sub.2CONH.sub.2
Glutamine 5 Phenylalanine 6 Tyrosine 7 Tryptophan --CH.sub.2SH
Cysteine --CH.sub.2CH.sub.2SCH.sub.3 Methionine --CH.sub.2OH Serine
--CH(OH)CH.sub.3 Threonine 8 Proline 9 Hydroxyproline
[0026] In addition, as used herein, an "amino acid side chain
derivative" represents modifications and/or variations to naturally
occurring amino acid side chain moieties. For example, the amino
acid side chain moieties of alanine, valine, leucine, isoleucine
and phenylalanine may generally be classified as alkyl, aryl, or
arylalkyl moieties, optionally substituted with one or more
substituents as defined below. Accordingly, representative amino
acid side chain derivatives include substituted or unsubstituted
alkyl, aryl and arylalkyl moieties.
[0027] To this end, "alkyl" is a straight chain or branched, cyclic
or noncyclic, saturated or unsaturated alkyl containing from 1 to
12 carbon atoms (also referred to herein as "C.sub.1-12alkyl").
Similarly, a "lower alkyl" is as defined above, but contains from 1
to 4 carbon atoms (also referred to herein as a "C.sub.1-4alkyl").
Representative saturated straight chain alkyls include methyl,
ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while
saturated branched alkyls include isopropyl, sec-butyl, isobutyl,
tert-butyl, isopentyl, and the like. Representative saturated
cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and the like. Unsaturated alkyls contain at least one
double or triple bond between adjacent carbon atoms (referred to as
an "alkenyl" or "alkynyl", respectively). Representative straight
chain and branched alkenyls include ethylenyl, propylenyl,
1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like; while representative straight chain and branched alkynyls
include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,
2-pentynyl, 3-methyl-1 butynyl, and the like. Representative
unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl,
and the like.
[0028] "Aryl" is an aromatic carbocyclic moiety contain from 6 to
12 carbon atoms (also referred to herein as a "C.sub.6-12aryl"),
such as phenyl and naphthyl.
[0029] "Arylalkyl" is an alkyl having at least one alkyl hydrogen
atom replaced with an aryl moiety, such as benzyl,
--(CH.sub.2).sub.2phenyl, --(CH.sub.2).sub.3phenyl,
--CH(phenyl).sub.2, and the like.
[0030] Similarly, the amino acid side chain moieties of histidine,
tryptophan, proline and hydroxyproline may generally be classified
as heterocyclic or heterocyclicalkyl moieties, optionally
substituted with one or more substituents as defined below.
Accordingly, representative amino acid side chain derivatives also
include substituted or unsubstituted heterocycle and
heterocyclealkyl moieties.
[0031] As used herein, "heterocycle" means a 5- to 7-membered
monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which
is either saturated, unsaturated, or aromatic, and which contains
from 1 to 4 heteroatoms independently selected from nitrogen,
oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms
may be optionally oxidized, and the nitrogen heteroatom may be
optionally quaternized, including bicyclic rings in which any of
the above heterocycles are fused to a benzene ring. The heterocycle
may be attached via any heteroatom or carbon atom. Heterocycles
include heteroaryls as defined below. Thus, in addition to the
heteroaryls listed below, heterocycles also include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl,
valerolactamyl, oxiranyl, oxetanyl, aziridinyl, azetidinyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
and the like.
[0032] "Heterocyclealkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heterocycle moiety, such as
--CH.sub.2(heterocycle), --(CH.sub.2).sub.2(heterocycle), and the
like.
[0033] "Heteroaryl" means an aromatic heterocycle ring of 5- to 10
members and having at least one heteroatom selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and bicyclic ring systems. Representative heteroaryls
are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,
quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl,
benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl.
[0034] "Heteroarylalkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heteroaryl moiety, such as
--CH.sub.2pyridinyl, --CH.sub.2pyrimidinyl, and the like.
[0035] The term "substituted" as used herein means any of the above
groups--that is, alkyl, aryl, arylalkyl, heterocycle,
heterocyclealkyl, heteroaryl or heteroarylalkyl--wherein at least
one hydrogen atom is replaced with a substituent. In the case of a
keto substituent ("C(.dbd.O)") two hydrogen atoms are replaced. A
"substituent" in this regard is halogen, keto, hydroxy, haloalkyl,
--R, --OR, --C(.dbd.O)R, --C(.dbd.O)OR, --C(.dbd.O)NRR, --NRR,
--NRC(.dbd.O)R, --NRC(.dbd.O)OR, --NRC(.dbd.O)NRR, --OC(.dbd.O)R,
--OC(.dbd.O)OR, --OC(.dbd.O)NRR, --SH, --SR, --SOR, --SO.sub.2R,
--NRSO.sub.2R, --SiR.sub.3, or --OP(OR).sub.3, wherein each
occurrence of R is the same or different and independently
hydrogen, alkyl, aryl, arylalkyl, heterocycle or heterocyclealkyl,
or wherein any two R groups attached to the same nitrogen atom,
taken together with the nitrogen atom to which they are attached,
form a heterocyclic ring or a substituted heterocyclic ring.
[0036] A "peptide" means at least two naturally or unnaturally
occurring alpha-amino acids joined via a peptide bond. Depending
upon the number of amino acids joined via peptide bonds, the
resulting peptide may also be referred to as a "polypeptide" or
"protein." Similarly, a "peptide derivative" means a peptide which
has been covalently modified and/or which contains amino acids
other than alpha-amino acids. Representative peptide derivatives
include peptides which are N-alkylated, N-acylated or
N-sulfonylated at the amino termini, with, for example, methyl,
benzyl, acetyl, benzoyl, methanesulfonyl, phenylsulfonyl,
allyloxycarbonyl, t-butyloxycarbonyl, benzyloxycarbonyl, or
fluorenyloxycarbonyl moieties; peptides in which the carboxy
termini are esterified (methyl, ethyl, benzyl) or reduced to a
hydroxy or aldehyde; peptides which are N-alkylated at peptide
bonds with, for example, methyl or 2-hydroxy-4-methoxybenzyl; and
peptides which incorporate beta- or gamma-amino acids such as
beta-alanine or gamma-aminobutyric acid.
[0037] A "linker" is any covalent bridging moiety that facilitates
linkage of a compound of structure (I), through the respective
R.sub.1, R.sub.2, R.sub.3, R.sub.4 and/or R.sub.5 moiety, to
another moiety, agent, compound, solid support, molecule, amino
acid, peptide or protein. For example, the compounds of this
invention may be linked to one or more known compounds, such as
biotin, for use in diagnostic or screening assays. Furthermore, one
(or more) of R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5 may be a
linker joining the compound of structure (I) to a solid support
(such as a support used in solid phase peptide synthesis). Examples
of such linkers include p-alkoxybenzyl alcohol,
phenylacetamidomethyl, and 2-chlorotrityl chloride. In this
context, linkage to another moiety or compound, or to a solid
support, is preferable at the R.sub.1 or R.sub.4 position.
[0038] A "solid support" means any composition of matter to which
another compound is attached directly or attached through a linker
and which is insoluble in at least one solvent that the attached
compound is soluble in. Alternatively, a "solid support" may be a
composition of matter with similar solubility characteristics to
the attached compound, but which may be readily precipitated from
solution and filtered off as a solid. Representative examples
include polystyrene, polyethylene glycol, polystyrene grafted with
polyethylene glycol, polyacrylamide, polyamide-polyethylene glycol
copolymer, controlled-pore glass, and silica.
[0039] The phrase "remainder of the molecule" means any moiety,
agent, compound, solid support, molecule, linker, amino acid,
peptide or protein covalently attached to the reverse-turn mimetic
at either the R.sub.1 and/or R.sub.4 positions, including amino
acid side chain moieties, amino acid side chain derivatives and
peptide derivatives as defined above. Accordingly, an alternative
depiction of structure (I), the bond between the ring nitrogen
atoms and the corresponding R.sub.1 and R.sub.4 moieties may be
left undefined, as represented by the following structure (I'):
10
[0040] wherein 11
[0041] represents the remainder of the molecule joined to the
corresponding ring nitrogen through a covalent bond, and A, R.sub.2
and R.sub.3 are as defined above.
[0042] In an embodiment of structure (I), R.sub.2a, R.sub.3a and
each occurrence of R.sub.5a and R.sub.5 are hydrogen, and the
compounds of this invention have the following structure (II):
12
[0043] wherein n, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as
defined above.
[0044] In another embodiment, A is --CH(R.sub.5)CH(R.sub.5)--,
R.sub.2a and R.sub.3a are both hydrogen, and the compounds of this
invention have the following structure (III): 13
[0045] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and each
occurrence of R.sub.5 are as defined above.
[0046] In still a further embodiment, n is 1, R.sub.2a, R.sub.3a
and R.sub.5a are hydrogen, and the compounds of this invention have
the following structure (IV): 14
[0047] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
as defined above.
[0048] In a more specific embodiment of structure (IV), R.sub.5 is
hydrogen and the compounds of this invention have the following
structure (V): 15
[0049] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined
above.
[0050] In yet another embodiment of structure (I), R.sub.3a is
hydrogen, and the compounds of this invention have the following
structure (VI): 16
[0051] wherein A, R.sub.1, R.sub.2, R.sub.2a, R.sub.3 and R.sub.4
are as defined above.
[0052] In a preferred embodiment of structure (I), R.sub.2a,
R.sub.3a and each occurrence of R.sub.5a are hydrogen, R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 are the same or different and
independently an amino acid side chain moiety or amino acid side
chain derivative. In a further preferred embodiment of structure
(I), R.sub.1 is --C(.dbd.O)OR.sub.7, --C(.dbd.O)NHR.sub.7 or
--SO.sub.2R.sub.7, where R.sub.7 is an amino acid side chain moiety
or an amino acid side chain derivative. In still a further
preferred embodiment of structure, R.sub.7 is aryl or arylalkyl
optionally substituted with halogen, --OH, --COOH, --NH.sub.2 or
C.sub.1-4alkyl.
[0053] In structure (I) above, a solid line designation for
attachment of the various R groups to a carbon atom on the fused
bicyclic ring indicates that these R groups may lie either above or
below the plane of the page. If a reverse-turn mimetic of this
invention is intended to mimic a reverse-turn of naturally
occurring amino acids (i.e., "L-amino acids"), the R groups would
generally lie below the plane of the page (i.e., "R") in Structure
(I). However, if the reverse-turn mimetic of this invention is
intended to mimic a reverse-turn containing one or more D-amino
acids, then the corresponding R group or groups would lie above the
plane of the page (i.e., "R") in Structure (I).
[0054] The reverse-turn mimetics of the present invention may
generally be prepared by the method illustrated in the following
Reaction Scheme. 17
[0055] In the above Reaction Scheme, a resin bearing free hydroxyl
groups is treated with a bromine-containing acetal under acidic
conditions. The derivatized resin is reacted with a primary amine
and subsequently acylated with an N-protected amino acid derivative
in the presence of a coupling reagent and base. The amino
protection is removed and a second N-protected amino acid
derivative is coupled. After deprotection, the exposed primary
amine is capped with a suitable reagent such as a sulfonyl
chloride, chloroformate, or isocyanate. The compound is
simultaneously removed from the resin and cyclized to form the
final product by treatment with formic acid. Alternatively, the
reverse-turn mimetics of structure (I) may be prepared in solution
by sequential or convergent coupling of the individual
components.
[0056] While the above Reaction Scheme depicts the R.sub.2a and
R.sub.3a moieties as hydrogen, compounds of structure (I) having
moieties other than hydrogen at the R.sub.2a and R.sub.3a position
may be made by the same Reaction Scheme, but using the
corresponding R.sub.2a-substituted and/or R.sub.3a-substituted
reaction precursors. For example, when R.sub.2 and R.sub.2a are
both methyl, a suitable aminoisobutyric acid derivative may be used
to introduce these groups into the reverse-turn mimetic.
[0057] As mentioned above, the reverse-turn mimetics of the present
invention are useful as bio-active agents, such as diagnostic,
prophylactic, and therapeutic agents. The integrin binding activity
of representative reverse-turn mimetics is presented in Example 2.
In this example, the reverse-turn mimetics were found to
effectively displace CS1 peptide from Ramos cells. The data thus
indicate the ability of reverse turn mimetics to antagonize
.alpha..sub.4.beta..sub.1 integrins and serve as potential
anti-inflammatory agents.
[0058] In another aspect of this invention, libraries containing
reverse-turn mimetics of the present invention are disclosed. Once
assembled, the libraries of the present invention may be screened
to identify individual members having bioactivity. Such screening
of the libraries for bioactive members may involve, for example,
evaluating the binding activity of the members of the library or
evaluating the effect the library members have on a functional
assay. Screening is normally accomplished by contacting the library
members (or a subset of library members) with a target of interest,
such as, for example, an antibody, enzyme, receptor or cell line.
Library members which are capable of interacting with the target of
interest are referred to herein as "bioactive library members" or
"bioactive mimetics". For example, a bioactive mimetic may be a
library member which is capable of binding to an antibody or
receptor, which is capable of inhibiting an enzyme, or which is
capable of eliciting or antagonizing a functional response
associated, for example, with a cell line. In other words, the
screening of the libraries of the present invention determines
which library members are capable of interacting with one or more
biological targets of interest. Furthermore, when interaction does
occur, the bioactive mimetic (or mimetics) may then be identified
from the library members. The identification of a single (or
limited number) of bioactive mimetic(s) from the library yields
reverse-turn mimetics which are themselves biologically active, and
thus useful as diagnostic, prophylactic or therapeutic agents, and
may further be used to significantly advance identification of lead
compounds in these fields.
[0059] Synthesis of the peptide mimetics of the library of the
present invention may be accomplished using known peptide synthesis
techniques, in combination with the component pieces of this
invention. More specifically, any amino acid sequence may be added
as any of the R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5
moieties of the conformationally constrained reverse-turn mimetic.
Preferably the amino acid sequence may be added as the R.sub.1 or
R.sub.4 moieties. To this end, the mimetics may be synthesized on a
solid support (such as polystyrene utilizing
4-hydroxymethylphenoxybutyrate as a linker) by known techniques
(see, e.g., John M. Stewart and Janis D. Young, Solid Phase Peptide
Synthesis, 1984, Pierce Chemical Comp., Rockford, Ill.; Atherton,
E., Shepard, R. C. Solid Phase Peptide Synthesis: A Practical
Approach; IRL: Oxford, 1989) or on a silyl-linked resin by alcohol
attachment (see Randolph et al., J. Am. Chem. Soc. 117:5712-14,
1995).
[0060] In addition, a combination of both solution and solid phase
synthesis techniques may be utilized to synthesize the peptide
mimetics of this invention. For example, a solid support may be
utilized to synthesize the linear peptide sequence up to the point
that the conformationally constrained reverse-turn is added to the
sequence. A suitable conformationally constrained reverse-turn
mimetic which has been previously synthesized by solution synthesis
techniques may then be added as the next "amino acid" to the solid
phase synthesis (i.e., the conformationally constrained
reverse-turn mimetic, which has at least two reactive sites, may be
utilized as the next residue to be added to the linear peptide).
Upon incorporation of the conformationally constrained reverse-turn
mimetic into the sequence, additional amino acids may then be added
to complete the peptide bound to the solid support. Alternatively,
the linear N-terminus and C-terminus protected peptide sequences
may be synthesized on a solid support, removed from the support,
and then coupled to the conformationally constrained reverse-turn
mimetic in solution using known solution coupling techniques.
[0061] In another aspect of this invention, methods for
constructing the libraries are disclosed. Traditional combinatorial
chemistry (see, e.g., The Combinatorial Index Bunin, Academic
Press, New York, 1998; Gallop et al., J. Med. Chem. 37:1233-1251,
1994) and parallel synthesis techniques permit a vast number of
compounds to be rapidly prepared by the sequential combination of
reagents to a basic molecular scaffold. For example, the above
disclosed synthesis may be carried out using the directed sorting
technique of Nicolaou and coworkers. (Nicolaou et al., Angew. Chem.
Int'l. Ed. 34:2289-2291, 1995). Presently, equipment for this
technique is commercially available from IRORI (La Jolla, Calif.).
Alternatively, the above disclosed synthesis may be carried out by
parallel synthesis using a 48- or 98-well plate format wherein each
well contains a fritted outlet for draining solvents and reagents
(A Practical Guide to Combinatorial Chemistry Czarnik and DeWitt,
Eds., American Chemical Society, Washington, DC, 1997). Robbins
(Sunnyvale, Calif.), Charybdis (Carlsbad, Calif.) and Bohdan
(Chicago, Ill.) presently offer suitable equipment for this
technique.
[0062] In a further aspect of this invention, methods for screening
the libraries for bioactivity and isolating bioactive library
members are disclosed. The libraries of the present invention may
be screened for bioactivity by a variety of techniques and methods.
Generally, the screening assay may be performed by (1) contacting a
library with a biological target of interest, such as a receptor,
and allowing binding to occur between the mimetics of the library
and the target, and (2) detecting the binding event by an
appropriate assay, such as by the calorimetric assay disclosed by
Lam et al. (Nature 354:82-84, 1991) or Griminski et al.
(Biotechnology 12:1008-1011, 1994). In a preferred embodiment, the
library members are in solution and the target is immobilized on a
solid phase. Alternatively, the library may be immobilized on a
solid phase and may be probed by contacting it with the target in
solution.
[0063] In another aspect, the present invention encompasses
pharmaceutical compositions prepared for storage or administration
which comprise a therapeutically effective amount of a compound of
the present invention in a pharmaceutically acceptable carrier or
diluent. Therapy with inhibitors of cell adhesion is indicated for
the treatment and prevention of a variety of inflammatory
conditions, particularly rheumatoid arthritis, inflammatory bowel
disease and asthma. Those experienced in this field are readily
aware of the circumstances requiring anti-inflammatory therapy.
[0064] The "therapeutically effective amount" of a compound of the
present invention will depend on the route of administration, the
type of warm-blooded animal being treated, and the physical
characteristics of the specific animal under consideration. These
factors and their relationship to determining this amount are well
known to skilled practitioners in the medical arts. This amount and
the method of administration can be tailored to achieve optimal
efficacy but will depend on such factors as weight, diet,
concurrent medication and other factors which as noted those
skilled in the medical arts will recognize.
[0065] The "therapeutically effective amount" of the compound of
the present invention can range broadly depending upon the desired
affects and the therapeutic indication. Typically, dosages will be
between about 0.01 mg/kg and 100 mg/kg body weight, preferably
between about 0.01 and 10 mg/kg, body weight.
[0066] "Pharmaceutically acceptable carriers" for therapeutic use,
including diluents, are well known in the pharmaceutical art, and
are described, for example, in Remingtons Pharmaceutical Sciences,
Mack Publishing Co. (Gennaro Ed. 1985). For example, sterile saline
and phosphate-buffered saline at physiological pH may be used.
Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. For example, sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. In addition, antioxidants and suspending
agents may be used.
[0067] Compounds of the present invention are useful for prevention
and treatment of any condition in which an excess of
integrin-mediated cell adhesion is a contributing factor. In
particular, the compounds of the present invention are useful as
agents for the prevention and treatment of inflammation. In the
practice of the methods of this invention, a composition containing
a therapeutically effective amount of a compound of this invention
is administered to a warm-blooded animal in need thereof. For
example, the compounds of this invention may be administered to a
warm-blooded animal that has been diagnosed with, or is at risk of
developing a condition selected from rheumatoid arthritis,
atherosclerosis, Alzheimer's disease, AIDS dementia, ARDS, asthma,
allergies, inflammatory bowel disease, CNS inflammation, atopic
dermatitis, type I diabetes, encephalitis, myocardial ischemia,
multiple sclerosis, meningitis, nephritis, reperfusion injury,
restenosis, retinitis, psoriasis, stroke and tumor metastasis.
[0068] Multiple sclerosis (MS) is a progressively debilitating
autoimmune disease of the central nervous system. Presently the
exact antigen triggering the immune response is unknown. However,
macrophages appear to attack and initiate the destruction of the
fatty myelin sheaths surrounding nerve fibers in the brain. In an
animal model of MS (experimental allergic encephalomyelitis) murine
monoclonal antibodies to .alpha..sub.4.beta..sub.1 blocked adhesion
of the leukocytes to the endothelium, and prevented inflammation of
the central nervous system and subsequent paralysis of the animals
(Yednock, Cannon et al., Nature 356: 63-6, 1992).
[0069] The compounds of the present invention may be used
singularly, as a combination of two or more compounds, or in
combination with other known inhibitors of inflammation. For
example the compounds of this invention may be used therapeutically
with corticosteroids, non-steroidal anti-inflammatory agents, COX-2
inhibitors, matrix metalloprotease inhibitors or lipoxygenase
inhibitors. The compounds of the invention can be administered in
such oral forms as tablets, capsules (each of which includes
sustained release or timed release formulations), pills, powders,
granules, elixers, tinctures, suspensions, syrups, and emulsions.
Likewise, they may be administered in intravenous (bolus or
infusion), intraperitoneal, subcutaneous, intranasal, intrarectal
or intramuscular form, all using forms well known to those of
ordinary skill in the pharmaceutical arts. The compounds may be
administered intraocularly or topically as well as orally or
parenterally.
[0070] The compounds of this invention may be administered by
inhalation, and thus may be delivered in the form of an aerosol
spray from pressurized packs or nebulizers. The compounds may also
be delivered as powders which may be formulated and the powder
composition may be inhaled with the aid of an insufflation powder
inhaler device. A preferred delivery system for inhalation is the
metered dose inhalation aerosol, which may be formulated as a
suspension or solution of a compound of the invention in suitable
propellants, such as fluorocarbons or hydrocarbons. Another
preferred delivery system is the dry powder inhalation aerosol,
which may be formulated as a dry powder of a compound of this
invention with or without additional excipients.
[0071] The compounds of the invention can be administered in the
form of a depot injection or implant preparation which may be
formulated in such a manner as to permit a sustained release of the
active ingredient. The active ingredient can be compressed into
pellets or small cylinders and implanted subcutaneously or
intramuscularly as depot injections or implants. Implants may
employ inert materials such as biodegradable polymers or synthetic
silicones, for example, Silastic, silicone rubber or other polymers
manufactured by the Dow-Corning Corporation.
[0072] The compounds of the invention can also be administered in
the form of liposome delivery systems, such as small unilamellar
vesicles, large unilamellar vesicles and multilamellar vesicles.
Liposomes can be formed from a variety of phospholipids, such as
cholesterol, stearylamine or phosphatidylcholines.
[0073] The compounds of this invention may also be delivered by the
use of monoclonal antibodies as individual carriers to which the
compound molecules are coupled. The integrin inhibitors may also be
coupled with soluble polymers as targetable drug carriers. Such
polymers can include polyvinlypyrrolidone, pyran copolymer,
polyhydroxy-propyl-methacrylamide-- phenol,
polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysi-
ne substituted with palmitoyl residues. Furthermore, the integrin
inhibitors may be coupled to a class of biodegradable polymers
useful in achieving controlled release of a drug, for example,
polylactic acid, polyglycolic acid, copolymers of polylactic and
polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric
acid, polyorthoesters, polyacetals, polydihydropyrans,
polycyanoacrylates and cross linked or amphipathic block copolymers
of hydrogels.
[0074] The dose and method of administration can be tailored to
achieve optimal efficacy but will depend on such factors as weight,
diet, concurrent medication and other factors which those skilled
in the medical arts will recognize. When administration is to be
parenteral, such as intravenous on a daily basis, injectable
pharmaceutical compositions can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions.
[0075] Tablets suitable for oral administration of active compounds
of the invention can be prepared as follows:
2 Amount-mg Active Compound 25.0 50.0 100.0 Microcrystalline
cellulose 37.25 100.0 200.0 Modified food corn starch 37.25 4.25
8.5 Magnesium stearate 0.50 0.75 1.5
[0076] All of the active compound, cellulose, and a portion of the
corn starch are mixed and granulated to 10% corn starch paste. The
resulting granulation is sieved, dried and blended with the
remainder of the corn starch and the magnesium stearate. The
resulting granulation is then compressed into tablets containing
25.0, 50.0, and 100.0 mg, respectively, of active ingredient per
tablet.
[0077] An intravenous dosage form of the above-indicated active
compounds may be prepared as follows:
3 Active Compound 0.5-10.0 mg Sodium Citrate 5-50 mg Citric Acid
1-15 mg Sodium Chloride 1-8 mg Water for Injection (USP) q.s. to 1
ml
[0078] Utilizing the above quantities, the active compound is
dissolved at room temperature in a previously prepared solution of
sodium chloride, citric acid, and sodium citrate in Water for
Injection (USP, see page 1636 of United States
Pharmacopoeia/National Formulary for 1995, published by United
States Pharmacopoeia Convention, Inc., Rockville, Md., copyright
1994).
[0079] The following examples are provided for purposes of
illustration, not limitation.
EXAMPLES
Example 1
[0080] Synthesis of Representative Compounds of Structure (I)
[0081] Synthesis of 2-bromo-1-ethoxy-ethyl-1-oxy-linked Resin (a)
18
[0082] In general, a batch of resin (ArgogelOH or hydroxymethyl
polystyrene) was refluxed in 1,2-dichloroethane (DCE) for 4 hours
in the presence of 8 equivalents of bromoalkylaldehyde diethyl
acetal and 2 equivalents of pyridinium p-toluenesulfonate (PPTS).
In one instance, hydroxymethyl polystyrene (10.0 g, 0.7 mmol OH/g,
7 mmol) and 3.5 g of PPTS (14 mmol) were suspended in 200 ml of
DCE. Then, a solution of 8.5 ml of 2-bromodiethoxyethane (ca. 56
mmol) in DCE (100 ml) was added with stirring and the reaction
mixture was heated at reflux (approx. 80.degree. C.). After 4 hours
the resin was filtered off and washed with 100 mL dimethylformamide
(DMF), 50 mL dimethylsulfoxide (DMSO), 100 mL DMF, 200 mL
dichloromethane (DCM), 50 mL 1,4-dioxane and finally with 100 mL
methanol. After drying, 11.73 g, of resin (a) was obtained. Bromine
analysis indicated quantitative loading.
[0083] Synthesis of Representative Compounds
[0084] Reactions were carried out in plastic disposable syringes of
the appropriate size, each fitted with a polypropylene frit to
retain the resin. After each step, resin batches were washed with
DMF (3.times.) and DCM (3.times.). Typically, a 0.03 mmol sample of
resin (a) (e.g., 50 mg of polystyrene resin with loading of 0.6
mmol Br/g), pre-swollen in DMF, was treated with 1 mL of a 2.0 M
solution of amine R.sub.4--NH.sub.2 (2 mmol) in DMSO at 60.degree.
C. for 16-24 hrs.
[0085] Next, the resin was reacted with 0.09 mmol of Fmoc amino
acid (FmocNH--CHR.sub.3--COOH) in the presence of HATU (34 mg, 0.09
mmol) and DIEA (0.032 ml, 0.18 mmol) in DMF (1 mL) until the
chloranil test was negative (typically 1-2 h). Subsequently, the
Fmoc protection was removed by treatment with a 25% (v/v)
piperidine/DMF solution (2 mL) over 20 min.
[0086] The resin was then reacted with 0.09 mmol of a second Fmoc
amino acid (FmocNH-CHR.sub.2-COOH) in the presence of DIC (0.014
ml, 0.09 mmol) and HOBt (14 mg, 0.09 mmol) in DMF (1 mL) until the
Kaiser test was negative (typically 1 hour). The resin was again
treated with 25% (v/v) piperidine/DMF solution (2 mL) over 20
min.
[0087] Finally, the resin-bound sequence was terminated by reaction
with sulfonyl chloride (R.sub.1SO.sub.2Cl, 0.3 mmol) in the
presence of DIEA (0.106 mL, 0.6 mmol) in DCM (1 mL) for 1 hr
(Kaiser test negative). Alternatively, chloroformate R.sub.1OCOCl
or isocyanate R1NCO (the latter does not require presence of DIEA)
was used instead of sulfonyl chloride for introduction of the
R.sub.1 moiety.
[0088] The washed and dried resin was re-swollen in DCM, drained
and treated with 1 mL of formic acid (96%) overnight at rt. In a
number of cases, an elevated temperature up to 60.degree. C. or an
extended reaction time was necessary to complete the cyclization
(for conditions see Table 2 below). The supernatant was collected
and combined with washes (2.times.0.5 mL of formic acid). The
residue obtained after evaporation of formic acid was redissolved
in acetonitrile/water 50:50 mixture, frozen and lyophilized. The
yields of crude material were 85-100%. The crude purity of
compounds bearing a sulfonyl moiety at R.sub.1 generally exceeded
80%.
[0089] Table 2 presents representative compounds of this invention
synthesized by the above procedure.
4TABLE 2 REPRESENTATIVE COMPOUNDS) 19 Cpd. No. R.sub.1 R.sub.2
R.sub.2a R.sub.3 R.sub.4 1 20 21 22 23 24 2 25 26 27 28 29 3 30 31
32 33 34 4 35 36 37 38 39 5 40 41 42 43 44 6 45 46 47 48 49 7 50 51
52 53 54 8 55 56 57 58 59 9 60 61 62 63 64 10 65 66 67 68 69 11 70
71 72 73 74 12 75 76 77 78 79 13 80 81 82 83 84 14 85 86 87 88 89
15 90 91 92 93 94 16 95 96 97 98 99 17 100 101 102 103 104 18 105
106 107 108 109 19 110 111 112 113 114 20 115 116 117 118 119 21
120 121 122 123 124 22 125 126 127 128 129 23 130 131 132 133 134
24 135 136 137 138 139 25 140 141 142 143 144 26 145 146 147 148
149 27 150 151 152 153 154 28 155 156 157 158 159 29 160 161 162
163 164 30 165 166 167 168 169 31 170 171 172 173 174 32 175 176
177 178 179 33 180 181 182 183 184 34 185 186 187 188 189 35 190
191 192 193 194 36 195 196 197 198 199 37 200 201 202 203 204 38
205 206 207 208 209 39 210 211 212 213 214 40 215 216 217 218 219
41 220 221 222 223 224 42 225 226 227 228 229 43 230 231 232 233
234 44 235 236 237 238 239 45 240 241 242 243 244 46 245 246 247
248 249 47 250 251 252 253 254 48 255 256 257 258 259 49 260 261
262 263 264 50 265 266 267 268 269 51 270 271 272 273 274 52 275
276 277 278 279 53 280 281 282 283 284 54 285 286 287 288 289 55
290 291 292 293 294
[0090]
5 Compound Cleavage LC RT.sup..dagger-dbl. MS No. Conditions (min)
(M + H.sup.+) 1 rt 3.56 (A) 465.5 2 rt 3.77 (A) 470.5 3 rt 4.01 (A)
538.6 4 rt 6.40 (A) 532.3 5 rt 7.26 (B) 568.3 6 rt 7.04 (B) 532.3 7
rt 7.78 (B) 568.3 8 40.degree. C. 2.64 (C) 580.5 9 40.degree. C.
2.58 (C) 564.4 10 40.degree. C. 2.67 (C) 550.4 11 40.degree. C.
2.27 (C) 468.4 12 rt 1.64 (D) 516.7 13 rt 1.54 (D) 520.8 14 rt 1.52
(D) 488.7 15 40.degree. C. 1.58 (D) 502.8 16 40.degree. C. 1.60 (D)
516.8 17 rt 1.45 (D) 478.3 18 rt 1.47 (D) 512.3 19 40.degree. C.
1.50 (D) 516.3 20 40.degree. C. 1.55 (D) 516.3 21 40.degree. C.
1.51 (D) 499.3 22 40.degree. C. 1.55 (D) 592.4 23 40.degree. C.
1.48 (D) 492.4 24 40.degree. C. 1.38 (D) 517.4 25 40.degree. C.
1.54 (D) 550.5 26 40.degree. C. 1.58 (D) 550.5 27 40.degree. C.
1.52 (D) 533.2 28 40.degree. C. 1.57 (D) 626.2 29 40.degree. C.
1.52 (D) 526.5 30 40.degree. C. 1.42 (D) 551.4 31 40.degree. C.
1.54 (D) 514.6 32 40.degree. C. 1.61 (D) 530.5 33 40.degree. C.
1.21 (D) 529.4 34 40.degree. C. 1.27 (D) 546.4 35 40.degree. C.
1.57 (D) 513.4 36 40.degree. C. (48 h) 1.62 (D) 530.5 37 40.degree.
C. 1.20 (D) 529.3 38 40.degree. C. 1.27 (D) 546.5 39 40.degree. C.
1.60 (D) 547.3 40 40.degree. C. 1.64 (D) 564.5 41 40.degree. C.
1.22 (D) 563.4 42 40.degree. C. 1.28 (D) 580.5 43 40.degree. C.
1.60 (D) 565.4 44 40.degree. C. 1.65 (D) 582.5 45 40.degree. C.
1.25 (D) 581.4 46 40.degree. C. 1.30 (D) 598.5 47 40.degree. C.
1.65 (D) 564.5 48 40.degree. C. 1.70 (D) 612.4 49 60.degree. C. (48
h) 1.47 (D) 599.4 50 40.degree. C. 1.53 (D) 676.5 51 40.degree. C.
1.68 (D) 612.4 52 40.degree. C. 1.71 (D) 612.4 53 40.degree. C.
1.65 (D) 612.4 54 40.degree. C. 1.40 (D) 614.3 55 40.degree. C.
1.70 (D) 578.3 .sup..dagger-dbl.LCMS analysis was performed on
reverse-phase C.sub.18 Zorbax columns using the following solvent
system: A, water with 0.1% formic acid; B, acetonitrile with 0.1%
formic acid. The following conditions were applied: (A), column 2.1
.times. 30 mm, 5-95% B in 4 min, flow 0.3 ml/min; (B), column 4.6
.times. 100 mm, 5-90% B in 15 min, flow 1.5 ml/min; (C), column 2.1
.times. 30 mm, 5-95% B in 3 min, flow 0.5 ml/min; (D), column 2.1
.times. 30 mm, 5-95% B in 2 min, flow 0.8 ml/min. # Mass spectra
for separated peaks were obtained either by electrospray (ES) or by
atmospheric pressure chemical ionization (APCI) using a MicroMass
LCZ mass spectrometer with the appropriate probes.
Example 2
[0091] Biological Activity of Representative Compounds
[0092] An assay measuring the ability of the compounds of Example 1
to antagonize binding of CS1 peptide to .alpha..sub.4.beta..sub.1
integrin was performed. A modification of the procedure of
Vanderslice, P. et al. (J. Immunol., 1997, 1710-1718) (incorporated
herein by reference) was utilized.
[0093] In brief, 100 .mu.L/well of a solution of biotinylated CS1
peptide (1 mg/100 mL of phosphate buffered saline (PBS)) was
incubated in a NeutrAvidin plate (Pierce) for 1 h at room
temperature. The plate was then washed 3.times. with distilled
water and treated with 200 .mu.L of blocking buffer (3% BSA in PBS)
for at least 4 h. Blocked plates were washed as above. Harvested
Ramos cells (10.sup.7/mL) were resuspended in PBS containing 10
.mu.L of calcein AM/mL and incubated 30 min in the dark. This
suspension was diluted with 45 mL PBS and the cells harvested by
centrifigation and aspiration. The cells were resuspended in
binding buffer (.about.5.times.10.sup.5/mL). If cell lysis was to
be monitored ethidium homodimer was added to the buffer to a final
concentration of 5 .mu.M. A solution (10 .mu.L) of compound to be
tested or control peptide was added to appropriate wells followed
by 90 .mu.L of the cell suspension. The plate was incubated at
37.degree. C. for 1 h. When ethidium homodimer was added,
fluorescence at 535/617 was measured before rinsing. Otherwise, the
plate was washed 3.times., 50 .mu.L of lysis buffer was added to
each well, the plate rocked in the dark for 10 min, and the
fluorescence monitored at 485 nm excitation and 535 nm
emission.
[0094] Preferably, the compounds of this invention have an
IC.sub.50 value of less than 100 .mu.M in this assay. To this end,
preferred compounds of this invention are compounds 4, 5, 8-10, 31,
32, 38-49, 54 and 55, and more preferred compounds having an
IC.sub.50 value of less than 10 .mu.M are compounds 10, 41, 42,
44-49, 54 and 55. As such, the compounds of this invention
effectively inhibit cell adhesion and possess activity as
anti-inflammatory agents.
[0095] It will be appreciated that, although specific embodiments
of the invention have been described herein for the purposes of
illustration, various modifications may be made without departing
from the spirit and scope of the invention. Accordingly, the
invention is not limited except by the appended claims.
* * * * *